title: Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering.
abstract: Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.
authors.affiliation: Key Laboratory of Systems Bioengineering, Tianjin University, Ministry of Education, Tianjin, 300072, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zha
authors.forename: Minghua
authors.initials: M
authors.lastname: Shen
authors.forename: Menglong
authors.initials: M
authors.lastname: Hu
authors.forename: Hao
authors.initials: H
authors.lastname: Song
authors.forename: Yingjin
authors.initials: Y
authors.lastname: Yuan
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D005075:Biological Evolution; D050542:D-Xylulose Reductase / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D009249:NADP / Q000378:metabolism; D010427:Pentose Phosphate Pathway / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015854:Up-Regulation; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
chemical_list: D000431:Ethanol; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase; D050542:D-Xylulose Reductase; D005947:Glucose; D014993:Xylitol